Our objective was to ascertain the frequency of antibodies to glutamic acid decarboxylase (GAD) in Europids and four Asian ethnic groups with insulin-dependent diabetes mellitus (IDDM) to gain insight into why the prevalence and incidence of IDDM varies so widely among ethnic and/or geographically diverse population groups. The subjects in this study were Europid (n = 49), Japanese (n = 16), Thai (n = 7), Korean (n = 21), and Chinese (n = 13) persons with IDDM with a duration ranging from 5 to 14 years. There were similar numbers of healthy controls matched for each ethnic group. A validated radioimmunoprecipitation assay used GAD from pig brain radiolabeled with 125I using chloramine T. Islet cell cytoplasmic antibodies measured by indirect immunofluorescence were expressed as Juvenile Diabetes Foundation units. The prevalence of antibodies to GAD, compared with Europids (63%), was much lower in all Asian populations with IDDM: Japanese (31%), Thai (29%), Korean (5%), and Chinese (27%). The mean level of antibodies to GAD, however, among diabetics from each population who gave a positive reaction, was similar. For all groups, the prevalence of antibodies to GAD was much higher than that of islet cell cytoplasmic antibodies. Almost all IDDM subjects positive for islet cell antibodies had antibodies to GAD, but the converse did not hold. A radioimmunoprecipitation assay for antibodies to GAD applied to serum from subjects with IDDM in various ethnic groups showed that Europids with IDDM had a much higher prevalence of such antibodies than did Asians. This held for all ethnic groups, and particularly Koreans. Thus, among different populations, there may be etiologic heterogeneity of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-Mr islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3–42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means ± 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.

Spectrin is the primary component of the erythrocyte membrane skeleton, and is composed of nine unique chemical domains and subdomains. This chapter presents an overview to develop procedures that allow the isolation of all nine structural domains and their subfragments from less than 20 ml of whole blood. The chapter presents a comparison between the structural and functional properties of individual spectrin domains from different donors. Preparative two-dimensional electrophoresis [isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS)] are used to resolve all peptides generated from spectrin by mild tryptic digestion. Following this, each domain can be identified by comparison to analytical two-dimensional separations of spectrin peptides and can be eluted rapidly from the gel. The most frequently used means of identifying spectrin peptides after two-dimensional electrophoresis is by immersing the gel in 1.0 M KCI at 0-40°. The amount of each peptide recovered ranges from 1 nm to several nanomoles. Each peptide can be compared from donor to donor using a variety of peptide mapping procedures.

Digestion of purified human erthrocyte spectrin with proteolytic enzymes at 0 degrees C results in the production of intermediate-size peptides that resist further cleavage at 0 degrees C. By two-dimensional peptide analysis of these intermediate peptides it has been determined that five unique peptides are produced by tryptic cleavage of the alpha subunit of spectrin (band 1); these have apparent molecular weights of 80,000, 46,000, 46,000, 41,000, and 30,000 and account for 97% of the alpha subunit. Similarly, four unique peptides having apparent molecular weights of 74,000, 65,000, 33,000, and 38,000 account for 90% of the beta subunit (band 2). By examining larger peptide fragments, the linear alignment of the unique peptides along each of the spectrin subunits has been established. These results indicate that spectrin is composed of two nonidentical subunits, each containing multiple proteolytically resistant domains. These domains, which may be largely alpha-helical, seem to be connected by small protease-sensitive segments. The proteolytic resistance of these domains is not influenced by the multimeric state of the spectrin molecule.